Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclofopmethyl-degrading biofilm consortium

Genomic DNA from nine individual bacteria, isolated from a diclofop-methyl-degrading biofilm consortium, was extracted for genetic characterization. The degradation of diclofop-methyl produces metabolites that are known intermediates or substrates for bacteria that degrade a variety of chlorinated aromatic compounds. Accordingly, oligonucleotide primers were designed from specific catabolic genes for chlorinated organic degradation pathways, and tested by PCR to determine if these genes are involved in diclofop-methyl degradation. DNA homology between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates, following 16S rDNA sequencing and identification of each of the isolates. These probes were successfully used for fluorescent in situ hybridization (FISH) studies of the two isolates in the biofilm consortium. Key words: consortium, catabolic gene, diclofop-methyl, 16S rDNA, FISH, SCLM.L\u2019ADN g\ue9nomique de neuf bact\ue9ries, isol\ue9es d\u2019un consortium d\u2019un biofilm d\ue9gradant le m\ue9thyldiclofop, a \ue9t\ue9 extrait pour fin de caract\ue9risation g\ue9n\ue9tique. Les m\ue9tabolites produits par le consortium lors de la d\ue9gradation du m\ue9thyldiclofop sont des interm\ue9diaires ou des substrats de bact\ue9ries d\ue9gradant des compos\ue9s chlor\ue9s. Cons\ue9quemment, des amorces oligonucl\ue9otidiques, sp\ue9cifiques pour des g\ue8nes cataboliques impliqu\ue9s dans la d\ue9gradation de compos\ue9s chlor\ue9s, ont \ue9t\ue9 test\ue9es par PCR pour d\ue9terminer l\u2019implication de ces g\ue8nes dans la d\ue9gradation de m\ue9thyldiclofop. L\u2019homologie existant entre les produits de PCR et les g\ue8nes \ue9tudi\ue9s, \ue9valu\ue9e par s\ue9quen\ue7age et transfert de Southern, sugg\ue8re l\u2019existence de nouveaux g\ue8nes cataboliques au sein du consortium. Suite au s\ue9quen\ue7age de l\u2019ADNr16S et \ue0 l\u2019identification des isolats, deux oligonucl\ue9otides fluorescents, sp\ue9cifiques pour deux isolats, ont \ue9t\ue9 utilis\ue9es avec succ\ue8s dans des \ue9tudes d\u2019hybridation in situ en fluorescence (FISH) de ces deux isolats du consortium dans le biofilm.NRC publication: Ye

Macrophage Bactericidal Activities against <i>Staphylococcus aureus</i> Are Enhanced <i>In Vivo</i> by Selenium Supplementation in a Dose-Dependent Manner

<div><p>Background</p><p>Dietary selenium is of fundamental importance to maintain optimal immune function and enhance immunity during infection. To this end, we examined the effect of selenium on macrophage bactericidal activities against <i>Staphylococcus aureus</i>.</p><p>Methods</p><p>Assays were performed in golden Syrian hamsters and peritoneal macrophages cultured with <i>S</i>. <i>aureus</i> and different concentrations of selenium.</p><p>Results</p><p>Infected and selenium-supplemented animals have significantly decreased levels of serum nitric oxide (NO) production when compared with infected but non-selenium-supplemented animals at day 7 post-infection (<i>p</i> < 0.05). A low dose of 5 ng/mL selenium induced a significant decrease in macrophage NO production, but significant increase in hydrogen peroxide (H₂O₂) levels (respectively, <i>p</i> = 0.009, <i>p</i> < 0.001). The NO production and H₂O₂ levels were significantly increased with increasing concentrations of selenium; the optimal macrophage activity levels were reached at 20 ng/mL. The concentration of 5 ng/mL of selenium induced a significant decrease in the bacterial arginase activity but a significant increase in the macrophage arginase activity. The dose of 20 ng/mL selenium induced a significant decrease of bacterial growth (<i>p</i> < 0.0001) and a significant increase in macrophage phagocytic activity, NO production/arginase balance and <i>S</i>. <i>aureus</i> killing (for all comparisons, <i>p</i> < 0.001).</p><p>Conclusions</p><p>Selenium acts in a dose-dependent manner on macrophage activation, phagocytosis and bacterial killing suggesting that inadequate doses may cause a loss of macrophage bactericidal activities and that selenium supplementation could enhance the <i>in vivo</i> control of immune response to <i>S</i>. <i>aureus</i>.</p></div